The proposed experiments are aimed at furthering our understanding of regeneration through knock- outs of particular pathways and combinations of pathways in mice. The purposes of our experiments are threefold: 1) to identify the isolation or interaction of pathways that may contribute to regeneration, 2) to provide indication for and identify potential side-effects of specific therapeutic targeting and their combinatorial targeting, and 3) to establish lines of mice with dramatically enhanced regeneration for further scientific research. The bases of these experiments come from 3 recent developments of observations of remarkable regeneration in mice or disinhibition in-culture: regeneration in PTEN mice, regeneration in PDE8 mice, and myelin- and Nogo-disinhibition of neurons lacking function of both Nogo co-receptors. Specifically in our experiments we will assess for regeneration in mice lacking both Nogo co-receptors, create and assess the regenerative ability of mice lacking PTEN and one of the Nogo co-receptors, and create triple knock-out mice lacking both Nogo co-receptors and PDE8 or PTEN, and assess for combinatorial effects on regeneration. By combining knock-outs of these mice, we will be able to assess these pathways for their combined involvement in regeneration after spinal cord injury (SCI), and it may result in producing more regeneration than was ever before possible. Additionally, we will characterize regeneration using a novel and powerful 3D technique that allows visualization of whole volumes of tissue without physical cutting, potentially revolutionizing the field for how regeneration is analyzed. Clinically, this work aims to accelerate the translation of basic science to therapeutic applications for SCI, potentially by identifying combinatorial therapeutic strategies, identifying new targets, and streamlining drug development by ruling out approaches that are found to not be promising. Furthermore, the pursuit of these projects and the use of these techniques will foster the scientific development of Mr. Rafer Willenberg, providing him excellent training for a future scientific career.